Plant-origin regulator protein and nucleic acid encoding the same

ABSTRACT

A regulator protein of a two-component signal transduction system in plants and a nucleic acid coding therefor are disclosed. The present invention provided an isolated protein having the amino acid sequence shown in SEQ ID NO:1 in the Sequence Listing or an amino acid sequence having a homology of not less than 30% to the amino acid sequence shown in SEQ ID NO:1, which functions as a regulator protein in a plant, and a nucleic acid coding therefor.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP97/03290, which has an Internationalfiling date of Sep. 18, 1997, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a regulator protein involved in a planttwo-component signal transduction system and to a nucleic acid codingfor the same.

BACKGROUND ART

Cytokinins are a group of plant hormones, and have important roles incontrolling growth, morphogenesis and flow of nutrients in plants.However, the mechanism by which a cytokinin is recognized by plant cellsand causes various actions was not known. As a cytokinin, zeatin iswell-known.

Inorganic nutrients including nitrogen largely influence on the growthand morphogenesis of plants. For example, in photosynthetic tissues ofmaize, enzymes related to C4 photosynthesis such as phosphoenolpyruvatecarboxylase (PEPC) are synthesized and accumulated depending on theamount of inorganic nitrogen absorbed from the roots (B. Sugiharto, K.Miyata, H. Nakamoto, H. Sasakawa, T. Sugiyam, Plant Physiol. 92:963,1990). Sugiharto et al. (B. Sugiharto, J. N. Burnell, T. Sugiyama, PlantPhysiol. 100:153, 1992) and Suzuki et al. (I. Suzuki, C. Cretin, T.Omata, T. Sugiyama, Plant Physiol. 105:1223, 1994) discovered that thissynthesis and accumulation are caused by the promotion of expression ofC4Ppc1, a gene encoding phosphoenolpyruvate carboxylase (PEPC), by acytokinin. Cytokinins are plant hormones synthesized in roots (L. J.Feldmn, in The Development and Function of Roots, J. G. Torrey and D. T.Clarkson Eds., Academic Press, London, 1975, pp.55-72), and thus it wasfound that they are important signal substances which transmit theinformation about the amount of the inorganic nitrogen, that was sensedby the roots.

When bacteria respond to various stimulations from outside, signaltransduction mechanism called two-component signal transduction systemplays an important role. It has become clear that the two-componentsignal transduction system is an important signal transduction systemalso in protozoa, fungi and plants (L. A. Alex and M. I. Simon, TrendsGenet. 10:133, 1992). The two-component signal transduction system iscomposed of a sensor protein which senses stimulations in the outside,and a regulator protein which mediates the signal and causes variousreactions. The signal transduction from the sensor protein to theregulator protein is mediated by phosphorylation reaction. The sensorprotein serves as a kinase specific to the regulator protein, and theregulator protein is modified by the phosphorylation. The regulatorprotein is activated by phosphorylation and causes specific geneexpression or the like. The sensor protein has a region in theN-terminal region, which senses the stimulation and a transmitter regionin the C-terminal region, that phosphorylates the regulator protein. Theregulator protein has a receiver region in the N-terminal region, whichis phosphorylated, and a region in the C-terminal region, that has anenzyme activity, DNA-binding activity or the like for causing variousreactions.

Known plant proteins related to the two-component signal transductionsystem include the following:

ETR1: This protein is a kind of receptor proteins of ethylene inArabidopsis. This protein is a kind of sensor proteins and has a regionhomologous to receiver regions of regulator proteins (C. Chang, S. K.Kwok, A. B. Bleecker, E. M. Meyerowits, Science 262:539-544, 1993).

ERS: This protein is a kind of ethylene receptor proteins of Arabidopsisand a kind of sensor proteins. Its ethylene-receiving region and thetransmitter region have high homologies to those of ETR 1. It does notcontain a region homologous to the receiver region (J. Hua, C. Chang, Q.Sun, E. M. Meyerowitz, Science 269:1712-1714, 1995).

NR: This protein is an ethylene receptor protein of tomato. This proteinhas a high homology to ETRI but does not have a region homologous to thereceiver region, like ERS (J. Wilkinson, M. B. Lanahan, H.-C. Yen, J. J.Giovannoni, H. J. Klee, Science 270:1807-1809, 1995).

CKI1: This protein is thought to be a cytokinin receptor protein ofArabidopsis and is a kind of sensor proteins. This protein has a regionhomologous to the receiver region like ETRI, in addition to thecytokinin receptor region and a transmitter region (T. Kakimoto, Science274:982-985, 1996).

Thus, those hitherto known in plants are sensor proteins similar to thesensor proteins of microorganisms or sensor proteins which have regionshomologous to the receiver region of a regulator protein. Thus, no plantregulator proteins are known.

Thus, it has been suggested that the signal transduction system inplants are composed of proteins and mechanisms having considerablydifferent properties from those involved in the signal transductionsystem of microoganisms.

On the other hand, if a protein or a DNA encoding a protein in a plantis known, a protein resulting from modification of the protein may beexpressed in plants, expression of the protein in the plant may beinhibited, the protein may be expressed in excess, or expression controlof the protein may be modified by using recombinant DNA technology andthe technology to construct transgenic plants.

Therefore, if a protein in a signal transduction system is known,manipulation of the signal tansduction system may be possible, and inturn, various life phenomena downstream thereof may be manipulated.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a regulator protein ina two-component signal trnasduction system of a plant and a nucleic acidcoding therefor.

The present inventors isolated a cDNA of a gene specifically induced bya cytokinin, from green leaves of maize in nitrogen-deficient state byusing differential display method; sequenced the cDNA and determined theamino acid sequence of the protein encoded by the cDNA; discovered thatthe amino acid sequence has a homology to regulator proteins ofmicroorganisms and that the amino acid sequence has characteristic aminoacids common to regulator proteins of microorganisms; and

confirmed that the gene is induced by nitrogen or a cytokinin; therebyconfirming that the protein is a regulator protein, to complete thepresent invention.

That is, the present invention provides an isolated protein having theamino acid sequence shown in SEQ ID NO:1 in the Sequence Listing or anamino acid sequence having a homology of not less than 30% to the aminoacid sequence shown in SEQ ID NO:1, which functions as a regulatorprotein in a plant. The present invention also provides an isolatednucleic acid encoding the protein of the present invention.

Only sensor proteins are known as proteins involved in two-componentsignal transduction systems of plant cells. In monocotyledons, even aprotein involved in a two-component signal transduction system is notknown. Thus, by the present invention, a regulator protein of atwo-component signal transduction system of plant cells and a nucleicacid coding therefor were first provided. Especially, a protein in atwo-component signal transduction system in monocotyledon was firstprovided by the present invention. Thus, by the present invention, thesignal transduction system of a cytokinin may be effectively modified bymanipulation of the two-component signal transduction system so as toeffectively control the various physiological mechanisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of CIP 1 (SEQ ID NO:1) which is anexample of the regulator protein according to the present invention incomparison with the amino acid sequence of CheY (SEQ ID NO:3) which is aregulator protein originated from E. coli and that of STYNTRrr (SEQ IDNO:4) which is a regulator protein originated from Salmonellatyphimurium.

BEST MODE FOR CARRYING OUT THE INVENTION

The nucleic acid according to the present invention, which codes for aregulator protein of a plant was obtained by the method detailed in theExamples below. Briefly, cDNA libraries were prepared from maize greenleaves treated with zeatin which is a kind of cytokinins and maize greenleaves not treated with zeatin, respectively. Using these cDNA librariesas templates, DNAs were amplified by PCR using random primers(radiolabelled dCTP was used so that amplification products areradiolabelled), and the amplification products were fractionated byelectrophoresis, followed by isolation of the cDNA from the gel, whichis expressed in the green leaves treated with zeatin but not expressedin the green leaves not treated with zeatin. Using this partial lengthcDNA as a probe, the cDNA library originated from the maize green leavestreated with zeatin was screened to obtain a full length cDNA cloneZmCip1.

The nucleotide sequence of ZmCip1 cDNA is shown in SEQ ID NO: 2 in theSequence Listing. It was proved that the nucleotide sequence of ZmCip1cDNA contains an open reading frame encoding a protein with a size of16.7 kDa having 157 amino acids, as shown in SEQ ID NO: 2. The aminoacid sequence alone shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1. Thisprotein was named CIP1 (abbreviation of cytokinin-induced protein).

The amino acid sequence of CIP1 has a high homology to the receiverregion of CheY (U.Kues, U. Stahl, Microbiol. Rev. 53:491, 1989) (FIG. 1)which is a kind of regulator proteins of microoganisms. CIP1 has theamino acid residues corresponding to Asp90 residue presumed as thephosphate binding site, and corresponding to Asp44 residue and Lys142residue in the active site, which amino acid residues are located in theregions in CheY, that have high homologies to other regulator proteins.CheY is the most analyzed regulator protein. The amino acid sequence ofCIP1 has a homology of 47% to that of CheY, and has a 42% homology tothe receiver region of CKI1 which is a cytokinin receptor protein ofArabidopsis. The N-terminal region of CIP 1 is unique to CIP1 and isconsiderably longer than that of CheY. The amino acid sequence of CIP1is shown in FIG. 1 in comparison with that of CheY from E. coli and thatof STYNTRrr from Salmonella typhimurium, which are regulator proteins ofmicroorganims.

As concretely described in the Examples below, it was experimentallyconfirmed that expression of CIP1 gene in plant leaves is induced by acytokinin and an inorganic nitrogen such as nitrate ion or ammonium ion,and is not induced in roots. From these data and the above-describedhomologies of the sequences, it was clarified that CIP1 encoded byZmCip1 cDNA is a regulator protein originated from a plant.

It is well-known in the art that there are cases wherein thephysiological activity of a physiologically active protein is retainedeven if one or more amino acids in the amino acid sequence aresubstituted or deleted, or even if one or more amino acids are added toor inserted into the amino acid sequence. Therefore, proteins having thesame amino acid sequence as shown in SEQ ID NO: 1 except that one ormore amino acid residues are substituted or deleted, or one or moreamino acid residues are added to or inserted in the amino acid sequence,which amino acid sequence has a homology of not less than 50% to thatshown in SEQ ID NO: 1, which proteins function as regulator proteins ina plant, as well as the nucleic acids coding for these proteins, arewithin the scope of the present invention. The above-mentioned homologyis preferably not less than 70%, more preferably not less than 80%, morepreferably not less than 90% and more preferably not less than 95%. Itis preferred that the aspartic acid residue which is the 44th amino acidresidue, the aspartic acid which is the 90th amino acid residue, and thelysine residue which is the 142nd amino acid residue in the sequenceshown in SEQ ID NO: 1 be conserved. Introduction of the above-mentionedsubstitution, deletion, insertion and addition of amino acid residues toan amino acid sequence of a protein may be attained by the well-knownsite-specific mutagenesis (Nucleic Acid Research, Vol. 10, No. 20,p6487-6500, 1982). By repeating the site-specific mutagenesis, it ispossible to construct a nucleic acid and protein having a relatively lowhomology.

The regulator protein according to the present invention is a regulatorprotein of a two-component signal transduction system involved in signaltransduction by a cytokinin in plant cells. Therefore, by expressing thegene of the regulator protein according to the present invention underan appropriate promoter as a sense or antisense gene by using therecombinant DNA technology, the technology to construct transgenicplants or the like, the signal transduction by a cytokinin may beturned-on and turned-off in a particular tissue at a particular time. Asa result, germination, growth, morphogenesis, photosynthesis,stress-resistance, flowering/fructification, dormancy, senescence or thelike may be controlled, so that novel varieties having highproductivity, high quality, stress-resistance and/or pest-resistance maybe developed. For example, since cytokinins have senescence-inhibitionactivities in plants, by expressing the gene of the regulator proteinaccording to the present invention under control of a promoter such asthe 35S promoter of cauliflower mosaic virus or the like, whichsystemically expresses genes in plants or a promoter which expressesgenes in senescence, the signal by a cytokinin may be transmitted inplant tissues even in senescence period, so that expression of the genesencoding proteins related to photosynthesis may be promoted or retained,whose expression is otherwise reduced in aging. Thus, by this method,novel varieties which attain increased biomass or yields may bedeveloped. Further, since cytokinins have an activity to initiate thegrowth of dormant buds of potato tubers, by expressing the gene of theregulator protein according to the present invention as an antisensegene under control of a promoter that expresses genes in stored tubersafter harvest so as to stop the transduction of the cytokinin signals, anovel potato variety in which germination of tubers is inhibited and dryweight loss is decreased, which may be stored for a long time, may becreated.

The invention will now be described more concretely by way of examples.

It should be noted that the present invention is not limited to theexamples below.

EXAMPLES

(1) Isolation of Partial Length cDNA Clone of Cytokinin-induced Gene byDifferential Display Method Maize (Zea mays L.cv. Golden Cross BantamT51) was cultivated in a growth chamber under nitrogen condition (0.8 mMNaNO₃) for 18 days as reported (B. Sugiharto, K. Miyata, H. Nakamoto, H.Sasakawa, T. Sugiyama, Plant Physiol. 92:963, 1990). The youngestexpanded leaves were cut at the distal end of the leaf blade in water byusing razorblade. Thereafter, the leaves were placed in a beakercontaining water with and without 5 μM t-zeatin, respectively, such thatthe distal ends of the leaves were downside. These leaves were placed inthe above-mentioned growth chamber for 40 minutes or 90 minutes.Thereafter, lower half of each leaf was collected and frozen with liquidnitrogen. From about 15 leaves, total RNAs were prepared by theguanidine thiocyanate/cesium chloride method.

Using a commercially available kit (RNAamp (trademark), Genhunter),differential display experiment (P. Liang, A. B. Pardee, Science257:967, 1992) was carried out for the obtained RNAs in accordance withthe instructions attached to the kit. Firstly, from the RNAs,single-stranded cDNA were synthesized by reverse transcription reactionusing oligo(dT) primer. The cDNAs were amplified by PCR using varioussynthetic primers (random primers). The reaction was carried out in thepresence of [α-³²P]dCTP to radiolabel the amplification product. Afterthe reaction, the PCR product was fractionated by 6% polyacrylamide gelelectrophoresis and autoradiography was carried out. The cDNA whoseexpression was observed to be induced by the 5 μM t-zeatin was isolatedfrom the gel and cloned into pT7Blue T-vector (trademark, Novagen). Bythese experiments, a partial length cDNA clone of a cytokinin-inducedgene was obtained.

(2) Isolation of Full Length cDNA Clone of Cytokinin-induced Gene andDetermination of DNA Sequence of the Same

Poly(A)⁺RNAs from the maize cut leaves treated with t-zeatin for 90minutes according to the method described above were prepared by aconventional method and a cDNA library was prepared using IMOSEIoxvector (trademark, Amersham).

Using the above-described partial length clone as a probe, the cDNAlibrary was screened to obtain a full length cDNA clone ZmCip1. DNAsequence of the ZmCip1 cDNA was determined by a conventional methodusing a commercially available DNA sequencer. The determined nucleotidesequence is shown in SEQ ID NO: 2 together with the deduced amino acidsequence encoded thereby.

(3) Assay of Induction of Expression of CIP 1 by Cytokinin Treatment(Run-Off Transcription Assay) Run-off transcription assay for expressionof ZmCip1 gene was carried out. Nuclei were isolated from maizemesophyll cells and its RNA synthesis activity was measured according toa reported method (I. Suzuki, C. Cretin, T. Omata, T. Sugiyama, PlantPhysiol.105:1223,1994). The RNAs synthesized by the isolated nuclei werefractionated by formaldehyde gel electrophoresis and the fractionatedRNAs were transferred to a nylon membrane, followed by hybridizationwith ³²P-labeled ZmCip 1 cDNA or C4Ppc1 cDNA. The intensity of thedetected signal was measured by Bio Imaging Analyzer (BAS2000, FujiFilm).

As a result, the treatment of the cut leaves of the maize cultivatedunder the low- nitrogen condition with t-zeatin for only 20 minutescauses transcription of ZmCip1 mRNA. This transcription occurred at muchearlier stage than the transcription of the mRNA of C4Ppc1 codingphosphoenolpyruvate carboxylase (PEPC) in the same leaves treated witht-zeatin.

(4) Analysis of Induction of Expression of CIP1 by Cytokinin Treatment(Northern Analysis)

The cut leaves of the maize cultivated under the low-nitrogen conditionas mentioned above was treated with 5 μM t-zeatin or with water for 0minute, 30 minutes, 45 minutes, 60 minutes or 90 minutes, and the ⅓portions from the distal ends of the leaves was collected. Afterfreezing the collected leaves with liquid nitrogen, RNAs were extractedby the guanidine thiocyanate/cesium chloride method. Ten micrograms ofthe obtained RNAs were fractionated by formaldehyde gel (1%)electrophoresis and the resultant was transferred to a nylon membrane.Hybridization was performed using ZmCip1 cDNA or maize ubiquitin gene asa probe.

As a result, accumulation of the mRNA of ZmCip1 was confirmed 30 minutesafter the cytokinin treatment in the nitrogen-deficient maize greenleaves and it was proved that the gene was strongly expressed 45-90minutes after the cytokinin treatment. In case where the cytokinintreatment was not performed, the expression was not detected.

(5) Analysis of Induction of Expression of CIP1 by Nitrate Ion (NorthernAnalysis)

The cut leaves of the maize cultivated under the low-nitrogen conditionas mentioned above were treated with 16 mM NaNO₃ solution or with 0.08mM NaNO₃ solution for 0 hour, 2 hours, 5 hours, 60 hours or 24 hours andNorthern analysis was carried out in the same manner as in (4) exceptthat the weight of the RNAs applied to each gel was 20 μg, respectively,and that a non-coding region of ZmCip1 was used as a probe.

As a result, accumulation of mRNA of ZmCip1 in the nitrogen-deficientgreen leaves was confirmed 5-6 hours after the nitrate ion treatment,and expression of the gene was scarcely observed 24 hours after thetreatment. In case where the nitrate ion treatment was not performed,the expression was not detected.

(6) Analysis of Induction of Expression of CIP1 by Nitrate Ion in Leavesand Roots (Northern Analysis)

The maize cultivated under the low-nitrogen condition as mentioned abovewas treated with 16 mM NaNO₃ solution or with 0.08 mM NaNO₃ solution for5 hours, and its leaf blades, leaf sheaths and roots were separatelycollected, followed by Northern analysis in the same manner as in (5)except that the amount of the RNAs from leaf sheaths or roots applied toeach gel was 30 μg.

As a result, accumulation of mRNA of ZmCip1 was confirmed 5 hours afterthe nitrate ion treatment in the nitrogen-deficient maize green leaves,while its expression was scarcely observed in roots.

(7) Analysis of Induction of Expression of CIP1 by Ammonium Ion(Northern Analysis)

The cut leaves of the maize cultivated under the low-nitrogen conditionas mentioned above were treated with 6 mM NH₄ ⁺ solution or with 0.08 mMNaNO₃ solution for 0 to 7 hours and Northern analysis was carried out inthe same manner as in (5) except that the weight of the RNAs applied toeach gel was 15 μg.

As a result, mRNA of ZmCip1 was confirmed 5 hours after the ammonium iontreatment in the nitrogen-deficient maize green leaves and it was provedthat the gene was strongly expressed 6 to 7 hours after the treatment.In case where the ammonium ion treatment was not performed, itsexpression was scarcely detected.

(8) Analysis of Induction of Expression of CIP1 by Cytokinin Treatmentand Cycloheximide Treatment (Northern Analysis)

The cut leaves of the maize cultivated under the low-nitrogen conditionas mentioned above were treated (i) with water for 60 minutes and thenwith water for 90 minutes, (ii) with water for 60 minutes and then with5 μM t-zeatin for 90 minutes, (iii) with 20 μg/ml of cycloheximide for60 minutes and then with 20 μg/ml cycloheximide and 5 μM t-zeatin for 90minutes, or (iv) with 20 μg/ml of cycloheximide for 60 minutes and thenwith 20 μg/ml of cycloheximide for 90 minutes, and Northern analysis wasperformed in the same manner as in (5) except that the amount of theRNAs applied to each gel was 30 μg.

As a result, mRNA of ZmCip 1 was confirmed 5 hours after the ammoniumion treatment in the nitrogen-deficient maize green leaves and it wasproved that the gene was strongly expressed 6 to 7 hours after thetreatment. In case where the ammonium ion treatment was not performed,its expression was scarcely detected.

(9) Analysis of Induction of Expression of CIP1 by Treatment with TraceAmount of Cytokinin (Northern Analysis)

The cut leaves of the maize cultivated under the low-nitrogen conditionas mentioned above were treated with 10⁻¹¹ to 10⁻⁷M t-zeatin solution orwater for 100 minutes or 180 minutes and then ⅓ portions from the distalends of the leaves were collected, followed by the Northern analysis asin (5).

As a result, it was confirmed that expression of mRNA of ZmCip1 wasinduced by the treatment with a very low level of t-zeatin as low as10⁻⁹ M.

(10) The cut leaves of the maize cultivated under the low-nitrogencondition as mentioned above were treated with 5 μM t-zeatin, 5 μMbenzyladenine, 10 μM±abscisic acid or with water for 100 minutes or 180minutes, and Northern analysis was performed as in (4).

As a result, it was proved that expression of mRNA of ZmCip1 was inducedin the nitrogen-deficient leaves by the benzyladenine treatment as thet-zeatin treatment, and that the expression was slightly induced by theabscisic acid treatment.

(11) Analysis of Induction of Expression of CIP1 by Sugar-boundCytokinin (Northern Analysis)

The cut leaves of the maize cultivated under the low-nitrogen conditionas mentioned above were treated with 5 μM t-zeatin, 5 μMzeatin-O-glucoside or with water for 100 minutes, and Northern analysiswas performed in the same manner as in (5), except that the amount ofthe RNAs applied to each gel was 15 μg.

As a result, it was proved that expression of mRNA of ZmCip1 was inducedin the nitrogen-deficient leaves by the zeatin-O-glucoside treatment asthe t-zeatin treatment.

Judging collectively from the results of (3) to (11), it is thought thatupon sensing nitrogen by the roots, a cytokinin is synthesized in theroots, the cytokinin is flowed to the leaves, expression of the CIP1protein is induced in the leaves and then expression of the genesrelated to C4 photosynthesis including C4Ppc1 is caused by thetwo-component signal transduction system involving the CIP1 protein.

4 1 157 PRT Maize 1 Met Ala Ala Ala Ala Pro Ala Pro Ala Ser Val Ala ProSer Ser Ala 1 5 10 15 Pro Lys Ala Thr Gly Asp Ser Arg Lys Thr Val ValSer Val Asp Ala 20 25 30 Ser Glu Leu Glu Lys His Val Leu Ala Val Asp AspSer Ser Val Asp 35 40 45 Arg Ala Val Ile Ala Arg Ile Leu Arg Gly Ser ArgTyr Arg Val Thr 50 55 60 Ala Val Glu Ser Ala Thr Arg Ala Leu Glu Leu LeuAla Leu Gly Leu 65 70 75 80 Leu Pro Asp Val Ser Met Ile Ile Thr Asp TyrTrp Met Pro Gly Met 85 90 95 Thr Gly Tyr Glu Leu Leu Lys Cys Val Lys GluSer Ala Ala Leu Arg 100 105 110 Gly Ile Pro Val Val Ile Met Ser Ser GluAsn Val Pro Thr Arg Ile 115 120 125 Thr Arg Cys Leu Glu Glu Gly Ala GluGly Phe Leu Leu Lys Pro Val 130 135 140 Arg Pro Ala Asp Val Ser Arg LeuCys Ser Arg Ile Arg 145 150 155 2 813 DNA Maize CDS (76)..(546) 2ctgtctgttt gcagcacccg caccacctga ctgtctgttt gcagcagccg cacctgtgtc 60ccgtccgtct ctgca atg gcc gct gcc gca ccg gct cca gca tct gtg gcg 111 MetAla Ala Ala Ala Pro Ala Pro Ala Ser Val Ala 1 5 10 ccg tcc tct gcg cccaag gcc acc ggc gac agc agg aag acg gtg gtg 159 Pro Ser Ser Ala Pro LysAla Thr Gly Asp Ser Arg Lys Thr Val Val 15 20 25 tcc gtg gac gcg tca gagctg gag aag cac gtg ctg gcg gtg gac gac 207 Ser Val Asp Ala Ser Glu LeuGlu Lys His Val Leu Ala Val Asp Asp 30 35 40 agc tcc gtg gac cgt gcc gtgatt gct agg atc ctg cgt ggc tcc agg 255 Ser Ser Val Asp Arg Ala Val IleAla Arg Ile Leu Arg Gly Ser Arg 45 50 55 60 tac agg gtg acc gcc gtg gagtcg gcg aca cga gcg ttg gag ctg ctc 303 Tyr Arg Val Thr Ala Val Glu SerAla Thr Arg Ala Leu Glu Leu Leu 65 70 75 gcg ctg ggc ctg ctt ccc gac gtcagt atg atc atc acc gac tac tgg 351 Ala Leu Gly Leu Leu Pro Asp Val SerMet Ile Ile Thr Asp Tyr Trp 80 85 90 atg ccc ggg atg acc ggg tac gag ctgctc aag tgc gtc aag gag tcg 399 Met Pro Gly Met Thr Gly Tyr Glu Leu LeuLys Cys Val Lys Glu Ser 95 100 105 gcg gcg cta agg ggc att ccc gtc gtcatc atg tcg tcg gag aac gtg 447 Ala Ala Leu Arg Gly Ile Pro Val Val IleMet Ser Ser Glu Asn Val 110 115 120 ccc acc cgt atc acc cgc tgc ctg gaggaa ggc gcc gag ggc ttc ctc 495 Pro Thr Arg Ile Thr Arg Cys Leu Glu GluGly Ala Glu Gly Phe Leu 125 130 135 140 ctc aag ccc gtc cgc ccc gcc gacgtc tcc cgc ctc tgc agc cgg atc 543 Leu Lys Pro Val Arg Pro Ala Asp ValSer Arg Leu Cys Ser Arg Ile 145 150 155 cgg tgactgcgtg tggtgctatgttaggagcta ggatcctcaa ccaaaaaaaa 596 Arg aagattcctc ttctttctttctttctctcc tgcttggaca tagatcttca aacaaggagc 656 taacatttgg ggggagactttttagcttta gggatctcaa caagctgttc ggaacggggg 716 ggatggagca cagcgttggctgttcttttc tccattcttc ttaataacat caggtgtcaa 776 tgtcatgcac gaaaaaaaaaaaaaaaaaaa aaaaaaa 813 3 132 PRT E.coli UNSURE (20)..(20) Xaa = anyamino acid, unknown or other 3 Met Ala Asp Lys Glu Leu Lys Phe Leu ValVal Asp Asp Phe Ser Thr 1 5 10 15 Met Arg Arg Xaa Ile Val Arg Asn LeuLeu Lys Glu Leu Gly Phe Asn 20 25 30 Asn Xaa Val Glu Glu Ala Glu Asp GlyVal Asp Ala Leu Asn Lys Leu 35 40 45 Gln Ala Gly Gly Tyr Gly Phe Val IleSer Asp Trp Asn Met Pro Asn 50 55 60 Met Asp Gly Leu Glu Leu Leu Lys ThrIle Arg Ala Asp Gly Ala Met 65 70 75 80 Ser Ala Leu Pro Xaa Val Leu MetVal Thr Ala Glu Ala Lys Lys Glu 85 90 95 Asn Ile Ile Ala Ala Ala Gln AlaGly Ala Ser Gly Tyr Val Val Lys 100 105 110 Pro Phe Thr Pro Ala Thr LeuGlu Glu Lys Leu Asn Lys Ile Phe Glu 115 120 125 Lys Leu Gly Met 130 4134 PRT S.typhimurium UNSURE (1)..(134) Any Xaa = any amino acid,unknown or other 4 Met Gln Arg Gly Ile Val Trp Val Val Asp Asp Asp SerSer Xaa Ile 1 5 10 15 Arg Trp Val Leu Glu Arg Ala Leu Ala Gly Ala GlyLeu Thr Cys Thr 20 25 30 Thr Phe Glu Asn Gly Asn Glu Val Leu Ala Ala LeuAla Xaa Ser Lys 35 40 45 Thr Pro Asp Val Xaa Leu Xaa Leu Ser Asp Ile ArgMet Pro Gly Met 50 55 60 Asp Gly Leu Ala Leu Leu Lys Gln Ile Lys Gln ArgHis Pro Met Xaa 65 70 75 80 Xaa Leu Pro Val Ile Ile Met Xaa Thr Ala HisSer Asp Leu Asp Ala 85 90 95 Ala Val Ser Ala Tyr Gln Gln Gly Ala Phe AspTyr Leu Pro Lys Pro 100 105 110 Phe Asp Ile Asp Glu Ala Val Ala Leu ValGlu Arg Ala Ile Ser His 115 120 125 Tyr Gln Glu Xaa Xaa Xaa 130

What is claimed is:
 1. An isolated protein comprising an amino acidsequence of SEQ ID NO:1 or an amino acid sequence having a sequenceidentity that is ≧70% to said SEQ ID NO:1, wherein said proteinfunctions as a regulator protein in a plant.
 2. The protein of claim 1,wherein the amino acid sequence is SEQ ID NO:1.
 3. The protein of claim1, wherein said protein is from a monocotyledon plant.
 4. An isolatednucleic acid coding for the protein of claim
 1. 5. The nucleic acid ofclaim 5, wherein the nucleotide sequence is SEQ ID NO:2.
 6. An isolatedprotein comprising a) a first amino acid sequence of SEQ ID NO:1 or b) asecond amino acid sequence having a sequence identity that is ≧80% tosaid SEQ ID NO:1, wherein the amino acid at position 44 of said secondamino acid sequence is aspartic acid, the amino acid at position 90 ofsaid second amino acid sequence is aspartic acid, and the amino acid atposition 142 of said second amino acid sequence is lysine, and whereinsaid protein comprising either first amino acid sequence or second aminoacid sequence functions as a regulator protein in a plant.
 7. Theprotein of claim 6, wherein said sequence identity is ≧90%.
 8. Theprotein of claim 6, wherein said sequence identity is ≧95%.
 9. Anisolated protein comprising a) a first amino acid sequence of SEQ IDNO:1 or b) a second amino acid sequence having 157 amino acid residueswith a sequence identity that is ≧70% to said SEQ ID NO:1, wherein saidprotein comprising either first amino acid sequence or second amino acidsequence functions as a regulator protein in a plant and is 16.7 kDa,and wherein the amino acid at position 44 of said second amino acidsequence is aspartic acid, the amino acid at position 90 of said secondamino acid sequence is aspartic acid, and the amino acid at position 142of said second amino acid sequence is lysine.